Leukotrienes and prostaglandins are inflammatory mediators important in asthma, arthritis, and other inflammatory diseases. Leukotrienes cause airway obstruction in asthmatics through bronchoconstriction, increased mucus secretion, and chemoattraction of inflammatory cells (O""Byrne, 1997); prostaglandins cause pain and edema associated with arthritis. Pharmacological intervention blocking either the synthesis or action of these lipid mediators is effective in treating human disease, thus confirming their importance (Simon et al., 1998; O""Byrne, 1997).
Cytosolic phospholipase A2 (cPLA2) initiates the production of leukotrienes and prostaglandins by releasing arachidonic acid from cellular membranes. Arachidonic acid in turn is metabolized to prostaglandins by the cyclooxygenase pathway and to leukotrienes by the 5-lipoxygenase pathway. Concomitant with the release of arachidonic acid, lyso-platelet-activating factor (lyso-PAF) is formed, which can then be acetylated to generate PAF, a molecule also implicated in the pathophysiology of asthma and arthritis (Venable et al., 1993). Hence, the reaction catalyzed by cPLA2 initiates the production of three classes of inflammatory mediators: leukotrienes, prostaglandins, and PAF.
cPLA2 is a member of a diverse superfamily of phospholipase A2 enzymes with the common ability to cleave the sn-2 ester of glycerophospholipids. The first members of the family to be characterized were the low molecular weight enzymes that are secreted either extracellularly or into granules (and are here collectively referred to as sPLA2s; groups I, II, III, V, VII, and IX) (Dennis, 1997). The PLA2 family has expanded with the cloning and characterization of calcium-dependent arachidonyl-selective cPLA2 (Clark et al., 1991; Kramer et al., 1991), the calcium-independent PLA2 (Tang et al., 1997; Balboa et al., 1997) and the plasma and intracellular PAF-acetylhydrolases (Hattori et al., 1994, 1995). Each of these new enzymes shares no sequence homology with the low molecular weight enzymes or with each other. In addition, unlike sPLA2s, which use activated water to cleave the phospholipid, these enzymes appear to use a nucleophilic serine. In this respect, they have more in common with other lipases of the xcex1/xcex2 hydrolase family than with the sPLA2s. Two additional enzymes with 30% identity to the catalytic domain of cPLA2 have recently been cloned; they have been termed cPLA2xcex2 (C. Song et al., manuscript in preparation) and cPLA2xcex3 (Underwood et al., 1998).
The cloning of cPLA2 is also described in U.S. Pat. Nos. 5,322,776, 5,354,677, 5,527698 and 5,593,878. The cloning of calcium-independent cPLA2 is also described in U.S. Pat. Nos. 5,466,595, 5,554,511, 5,589,170 and 5,840,511.
Numerous pieces of evidence have supported the central role of cPLA2 in lipid mediator biosynthesis. cPLA2 is the only enzyme which is highly selective for phospholipids containing arachidonic acid in the sn-2 position (Clark et al., 1995; Hanel and Gelb, 1993). Activation of cPLA2 or its increased expression have been linked with increased leukotriene and prostaglandin synthesis (Lin et al., 1992b). Following activation, cPLA2 translocates to the nuclear membrane, where it is co-localized with the cyclooxygenase and lipoxygenase that metabolize arachidonate to prostaglandins and leukotrienes (Schievella et al., Glover et al., 1995). Although these data are compelling, the most definitive evidence for the central role of cPLA2 in eicosanoid and PAF production came from mice made deficient in cPLA2 through homologous recombination (Uozumi et al., 1997; Bonventre et al., 1997). Peritoneal macrophages derived from these animals failed to make leukotrienes, prostaglandins, or PAF. The cPLA2 deficient mice have also been informative of the role of cPLA2 in disease, since these mice are resistant to bronchial hyperreactivity in an anaphylaxis model used to mimic asthma (Uozumi et al., 1997).
cPLA2 consists of at least two functionally distinct domains: a N-terminal Ca2+-dependent lipid-binding (CaLB) domain and a Ca2+-independent catalytic domain (Nalefski et al., 1994). The N-terminal CaLB domain is a member of the C2 family and its structure has been solved (Perisic et al., 1998; Xu et al., 1998); it mediates calcium regulation by co-localizing the catalytic domain with its membrane substrate (Nalefski et al., 1994). cPLA2 activity, in addition, is also regulated by phosphorylation of the catalytic domain (Lin et al., 1991; Leslie, 1997). Ser505 (of SEQ ID NO:2) and Ser727 (of SEQ ID NO:2) are conserved across all species and are phosphorylated in multiple cell types (de Carvalho et al., 1998). Phosphorylation of Ser505 (of SEQ ID NO:2) by members of the MAP-kinase family is a common response to extracellular stimuli that release arachidonic acid. Mutation of Ser505 (of SEQ ID NO:2) to Ala decreases activation (Lin et al., 1993) whereas the analogous mutation on Ser727 (of SEQ ID NO:2) has no effect (Leslie, 1998).
Several lines of evidence suggest that the catalytic mechanism of cPLA2 proceeds through a serine-acyl intermediate (Trimble et al., 1993; Hanel and Gelb, 1995). Mutation of Ser228 (of SEQ ID NO:2) abolishes cPLA2 activity against all substrates including phospholipids, lysophospholipids, and fatty acylated coumarin (Pickard et al., 1996; Huang et al., 1996). Ser228 (of SEQ ID NO:2) is present in a pentapeptide sequence, G-L-S-G-S (SEQ ID NO:3), which is similar to the classic xe2x80x9clipase motifxe2x80x9d G-X-S-X-G (Schrag and Cygler, 1997) found in most lipases within the broader family of enzymes called the xcex1/xcex2 hydrolases. These enzymes possess a common core which consists of a well-conserved mixed xcex2 sheet whose strands are interspersed by xcex1 helices. In all xcex1/xcex2 hydrolases, the catalytic serine is present in a tight turn between a xcex2-strand and an xcex1-helix, termed the xe2x80x9cnucleophilic elbowxe2x80x9d (see review by Schrag and Cygler, 1997). This turn directs the short serine side chain away from the protein backbone, reducing the steric hindrance about the residue and requiring that the +2 and xe2x88x922 sidechains be small to avoid steric clash; thus the prevalence of the G-X-S-X-G motif (Derewenda and Derewenda, 1991).
In addition to serine, xcex1/xcex2 hydrolases use a histidine and an acid (aspartate/glutamate) as the other members of a catalytic triad simliar to that present in serine proteases (Schrag and Cygler, 1997). However, although in cPLA2 Asp549 (of SEQ ID NO:2) was shown to be essential for activity, none of the 19 histidine residues were (Pickard et al., 1996). A different residue, Arg200 (of SEQ ID NO:2), was implicated as playing a role in the enzymatic process, although the mechanism for its involvement remained unknown. These observations suggested that cPLA2 acts through a novel catalytic mechanism for acyl hydrolases.
Like both the sPLA2s and the lipases of the xcex1/xcex2 hydrolase family, cPLA2 preferentially cleaves substrates presented in an interface (Nalefski et al., 1994). This phenomenon, known as interfacial activation, has been attributed to either conformational changes in the enzyme or more favorable presentation of the substrate (Scott et al., 1990). The origin of the 1500-fold difference in cPLA2 activity toward monomeric and micellar substrate remains unknown.
Despite the key role of cPLA2 in inflammatory disease, its three-dimensional structure remained unsolved, leaving numerous questions unanswered. Here we report the x-ray crystal structure of human cPLA2 at 2.5 xc3x85 resolution. The structure provides insight into the origin of arachidonate selectivity and interfacial activation, clarifies the roles of Ser228, Asp549, and Arg200 (of SEQ ID NO:2), and reveals the interplay between CaLB and the catalytic domains. Importantly, the structure is of a unique topology, distinct from that of the xcex1/xcex2 hydrolase family.
All references to amino acids in cPLA2 herein are made using residue numbers which refer to the cPLA2 sequence found in SEQ ID NO:2 and in Table I of U.S. Pat. No. 5,527,698, with the first methionine being designated residue 1 (Met1). SEQ ID NO:2 is encoded by the nucleotide sequence set forth in SEQ ID NO:1.
The present invention provides for crystalline cPLA2. Preferably, the cPLA2 is either human cPLA2 or cPLA2 from a non-mammalian species. In certain embodiments, the cPLA2 is recombinant cPLA2 and/or comprises the mature sequence of naturally-occurring cPLA2.
Other embodiments provide for a crystalline composition comprising cPLA2 in association with a second chemical species. Preferably, the second chemical species is selected from the group consisting of a potential inhibitor of cPLA2 activity and a potential inhibitor of cPLA2 membrane binding.
Yet other embodiments provide for a model of the structure of cPLA2 comprising a data set embodying the structure of cPLA2. Preferably, such data set was determined by crystallographic analysis of cPLA2, including possibly by NMR analysis. In certain embodiments, the data set embodies a portion of the structure of cPLA2, including without limitation the active site of cPLA2 or the CaLB domain of cPLA2.
Any available method may be used to construct such model from the crystallographic and/or NMR data disclosed herein or obtained from independent analysis of crystalline cPLA2. Such a model can be constructed from available analytical data points using known software packages such as HKL, MOSFILM, XDS, CCP4, SHARP, PHASES, HEAVY, XPLOR, TNT, NMRCOMPASS, NMRPIPE, DIANA, NMRDRAW, FELIX, VNMR, MADIGRAS, QUANTA, BUSTER, SOLVE, O, FRODO, RASMOL, and CHAIN. The model constructed from these data can then be visualized using available systems, including, for example, Silicon Graphics, Evans and Sutherland, SUN, Hewlett Packard, Apple Macintosh, DEC, IBM, and Compaq. The present invention also provides for a computer system which comprises the model of the invention and hardware used for construction, processing and/or visualization of the model of the invention.
Further embodiments provide a computer system comprising computer hardware and the model of the present invention.
Methods are also provided for identifying a species which is an agonist or antagonist of cPLA2 activity or binding comprising: (a) providing the model of the present invention, (b) studying the interaction of candidate species with such model, and (c) selecting a species which is predicted to act as said agonist or antagonist. Species identified in accordance with such methods are also provided.
Other embodiments provide: (1) a process of identifying a substance that inhibits cPLA2 activity or binding comprising determining the interaction between a candidate substance and a model of the structure of cPLA2, or (2) a process of identifying a substance that mimics cPLA2 activity or binding comprising determining the interaction between a candidate substance and a model of the structure of cPLA2. Substances identified in accordance with such processes are also provided.
The study of the interaction of the candidate species with the model can be performed using available software platforms, including QUANTA, RASMOL, O, CHAIN, FRODO, INSIGHT, DOCK, MCSS/HOOK, CHARMM, LEAPFROG, CAVEAT(UC Berkley), CAVEAT(MSI), MODELLER, CATALYST, and ISIS.
Other embodiments provide a method of identifying inhibitors of cPLA2 activity by rational drug design comprising: (a) designing a potential inhibitor that will form non-covalent bonds with one or more amino acids in the cPLA2 active site based upon the crystal structure co-ordinates of cPLA2; (b) synthesizing the inhibitor; and (c) determining whether the potential inhibitor inhibits the activity of cPLA2. Preferably, the crystal structure co-ordinates of cPLA2 used in such methods are obtained from a cPLA2 crystal of space group P21212 with a=153.59 angstroms, b=95.49 angstroms, and c=139.13 angstroms. In other preferred embodiments, the inhibitor is designed to interact with one or more atoms of said one or more amino acids in the cPLA2 active site is selected from the group consisting of:
CB and Oxcex3 atoms of Ser228 of SEQ ID NO:2;
Oxcex41 and O xcex42 atoms of Asp549 and Asp575 of SEQ ID NO:2;
CB, CG, CD, NE, CZ, NH1 and NH2 atoms of Arg200, Arg413 and Arg579 of SEQ ID NO:2;
Backbone carbonyl oxygen of Trp393 of SEQ ID NO:2;
Nxcex42 and Oxcex41 atoms of Asn555 of SEQ ID NO:2;
Atoms CD1, CE1, CG, CZ, CE2, and CD2 of Phe397, Phe681, Phe683 and Phe199 of SEQ ID NO:2;
CG, CD1, NE1, CE2, CZ2, CH2, CZ3, CE3 and CD2 of Trp232 and Trp393 of SEQ ID NO:2;
CB and Oxcex3 atoms of Ser577 of SEQ ID NO:2;
Atom s CB and Sxcex3 of Cys331 of SEQ ID NO:2;
Atoms OE1 and OE2 of Glu589 of SEQ ID NO:2;
Atoms CB, CG, CD, CE and NZ of Lys588 of SEQ ID NO:2;
Oxcex31 atom of Thr680 of SEQ ID NO:2;
OE1 and OE2 atoms of Glu418 and Glu422 of SEQ ID NO:2;
Atoms CB, CG, SD and CE of Met417 of SEQ ID NO:2;
Atoms CB, CG, CD1 and CD2 of Leu400 and Leu421 of SEQ ID NO:2;
Atoms CB, CG1, CG2, or CD1 of Ile424 of SEQ ID NO:2;
Backbone NH and carbonyl oxygen atoms of Ala578 of SEQ ID NO:2; and
Atoms CB, CG, ND1, CE1, NE2, and CD2 of His639 of SEQ ID NO:2.
Agonists and antagonists identified by such methods are also provided.
Methods are also provided for identifying inhibitors of cPLA2 membrane binding by rational drug design comprising: (a) designing a potential inhibitor that will form non-covalent bonds with one or more amino acids in the cPLA2 electrostatic patch region based upon the crystal structure co-ordinates of cPLA2; (b) synthesizing the inhibitor; and (c) determining whether the potential inhibitor inhibits the membrane binding of cPLA2. Preferably, the crystal structure co-ordinates of cPLA2 used in such methods are obtained from a cPLA2 crystal of space group P21212 with a=153.59 angstroms, b=95.49 angstroms, and c=139.13 angstroms. In other preferred embodiments, the inhibitor is designed to interact with one or more amino acids selected from the group consisting of Arg467, is Arg485, Lys488, Lys544 and Lys543 (all of SEQ ID NO:2). Agonists and antagonists identified by such methods are also provided.